Recently, it is particularly highly needed to rapidly, simply, and highly sensitively perform a quantitative measurement on microorganisms which may cause food poisoning or an infectious disease and which may cause any harm to the human body, because, in a step of producing food, a clinic unequipped with a microorganisms test facility, or the like, when a microorganisms test is performed on the spot, it is possible to prevent occurrence of food poisoning or an infectious disease.
In a so-called bio-sensor, when a biochemical substance in a sample is to be quantitatively measured by using artificial microspheres such as polystyrene labeled with a substance which is to be uniquely bonded to the measuring object, such as an antibody, it is necessary to quantitatively measure the number of microspheres in the sample or their bonding state. As described above, today, it is highly requested to rapidly, simply, and highly sensitively perform a quantitative measurement on microspheres contained in a liquid.
Here, the definition of the microspheres in the application will be described. The microspheres in the application are such as: polystyrene and the like substances, and particles in which any coating is applied to the substance; carbon nanotubes; metal particles such as gold colloids; and a living body or microspheres derived from a living body in a broad sense including small ones of so-called microorganisms, protozoans, and protozoas classified as a bacterium, a fungus, an actinomycete, a rickettsia, mycoplasma, or virus, larvae of organisms, cells of animals and plants, sperms, blood cells, nuclei acid, proteins, and the like. In addition, the microspheres in the application mean all particles which can be manipulated by dielectrophoresis. In the application, particularly, a measurement on microorganisms is assumed.
As the detection object in the application, particularly, microorganisms contained in blood and saliva of a human or an animal, or those collected from the surface of mucosa or the like to which blood or saliva of a human or an animal adheres are assumed. In blood and saliva of a human or an animal, ions are contained in high concentrations. This incurs a rise and variation of the electric conductivity of sample solutions, and causes a variation factor of a detecting method using dielectrophoresis.
Conventionally, the most widely used method of testing microorganisms is the culture method. In the culture method, a sample of microorganisms is smeared on a culture medium, the cultivation is performed under growth conditions for the microorganisms, and the number of colonies formed on the culture medium is counted, thereby quantitating the number of the microorganisms.
However, the colonization requires usually one to two days, or several weeks depending on the kind of microorganisms, and hence there is a problem in that the test cannot be rapidly performed. Furthermore, operations such as concentration, dilution, and smearing on the culture medium are necessary. Such operations must be performed by an expert. Consequently, there are problems in that the test cannot be simply performed, and that the accuracy is lowered by operational variations.
In order to solve the conventional problems, the inventor and other inventors have proposed a DEPIM (Dielectrophoretic Impedance Measurement) method in which dielectrophoresis and an impedance measurement are combined, as a rapid, simple, and highly sensitive method of counting the number of microorganisms (for example, see Patent Document 1).
In the DEPIM method, microorganisms are collected on microelectrodes by a dielectrophoretic force, and simultaneously an impedance change of the microelectrodes is measured, thereby quantitatively measuring the number of microorganisms in a sample liquid. Hereinafter, the measurement principle will be briefly described.
Usually, microorganisms have a structure where a cytoplasm which is ion-rich and high in dielectric constant and electric conductivity is surrounded by a cell membrane and cell wall which are relatively low in dielectric constant and electric conductivity, and can be deemed as dielectric particles. In the DEPIM method, a dielectrophoretic force which is a force acting, in a constant direction, on dielectric particles that are polarized in an electric field is used, and microorganisms which are dielectric particles are collected in the gap between the microelectrodes.
It is known that a dielectrophoretic force FDEP which acts on dielectric particles is given by following (Mathematical Formula. 1) (for example, see Non-patent Document 1). Hereinafter, description will be made while taking the case where dielectric particles are microorganisms, as an example.FDEP=2πa3∈0∈mRe[K]∇E2  [Mathematical Formula 1]
where a: the radius of a microorganism in case of sphere approximation, ∈0: dielectric constant in vacuum, ∈m: relative dielectric constant of a sample liquid, and E: electric field intensity, and ∇ is an operator indicating the gradient. In this case, ∇E2 shows the gradient of the electric field E2, and means the degree of inclination of E2 at the position, i.e., how steeply the electric field E spatially changes. Furthermore, K is called the Clausius-Mossoti factor, and indicated by (Mathematical Formula 2), and Re[K]>0 indicates positive dielectrophoresis in which the microorganisms move toward the high electric field region. Re[K]<0 indicates negative dielectrophoresis in which the microorganisms repelled from the high electric field region.
                    K        =                                            ɛ              b              *                        -                          ɛ              m              *                                                          ɛ              b              *                        +                          2              ⁢                              ɛ                m                *                                                                        [                  Mathematical          ⁢                                          ⁢          Formula          ⁢                                          ⁢          2                ]            
where ∈b* and ∈m* indicate the complex dielectric constants of the microorganisms and a solution, respectively. Usually, a complex dielectric constant ∈r* is indicated by (Mathematical Formula 3).
                              ɛ          r          *                =                              ɛ            r                    -                      j            ⁢                          σ                              ωɛ                0                                                                        [                  Mathematical          ⁢                                          ⁢          Formula          ⁢                                          ⁢          3                ]            
where ∈r: relative dielectric constant of the microorganisms or the sample liquid, σ: electric conductivity of the microorganisms or the sample liquid, and ω: angular frequency of the applied electric field.
From (Mathematical Formula 1), (Mathematical Formula 2), and (Mathematical Formula 3), it is seen that the dielectrophoretic force depends on the radius of a microorganism, the real part (hereinafter, indicated as Re[K]) of the Clausius-Mossoti factor, and the electric field intensity. Furthermore, it is seen that Re[K] is changed in dependence on the complex dielectric constants of the sample liquid and the microorganisms, and the frequency of the electric field.
In the DEPIM method, therefore, these parameters must be adequately selected, so that the dielectrophoretic force acting on the microorganisms is made sufficiently large and the microorganisms are surely collected in the electrode gap. The DEPIM method is characterized in that the electrical measurement is performed simultaneously with the microorganism collection to the electrodes by the dielectrophoresis, thereby quantitatively measuring the number of microorganisms in the sample liquid.
A microorganism has the above-described structure, and hence can be deemed as a microspheres which has electrically a specific impedance. When the number of microorganisms which are collected in the gap between the microelectrodes by dielectrophoresis is increased, therefore, the impedance between the electrodes is changed in accordance with the number of collected microorganisms.
Therefore, the inclination of a time change of the inter-electrode impedance has a value according to the number of microorganisms which are collected in the electrode gap per unit time, and the degree of the inclination corresponds to the concentration of the microorganisms in the sample liquid. When the inclination of a time change of the inter-electrode impedance is measured, consequently, it is possible to measure the concentration of the microorganisms in the sample liquid, or in other words the number of the microorganisms.
In the DEPIM method, furthermore, the number of the microorganisms is quantitated from the inclination of a time change of the impedance immediately after the start of dielectrophoresis, whereby the measurement of microorganisms is realized for a short time. In the above, the measurement principle of the DEPIM method has been briefly described. For details of the principle, please refer Non-patent Document 2.
The sample liquid which is used in the measurement in the application is assumed to be a liquid in which microorganisms collected by any method, such as blood or saliva is suspended in a liquid of a low electric conductivity and containing water as the principal constituent. It is seemed that, when microorganisms are collected, not only microorganisms but also ions contained in the vicinity are simultaneously collected. In this case, the dielectric constant of the sample liquid has a value which is substantially equal to that of water, with the result that the dielectrophoretic force acting on the microorganisms depends on the ion concentration of the sample liquid, or in other words the electric conductivity.
As the electric conductivity of a sample liquid is higher, usually, the dielectrophoretic force is smaller. In the case where it is assumed that a sample liquid such as described above is measured by the conventional DEPIM method, therefore, there is a problem in that, in a sample which has a high sample liquid electric conductivity, the dielectrophoretic force acting on microorganisms is reduced, and the number of microorganisms collected on microelectrodes is decreased, with the result that the measurement sensitivity is lowered. Moreover, the dielectrophoretic force acting on microorganisms is different depending on the sample liquid electric conductivity, and hence there is a problem in that, when sample liquids of different electric conductivities are measured, the dispersion of measurement results is large.
As means for solving the problems in a measurement of microorganisms or the like using dielectrophoresis, a technique is known in which, before a measurement, the sample liquid electric conductivity is reduced by ion exchange or the like. The technique is a method in which, before analysis, a sample liquid is treated in an ion-exchange column to reduce the sample electric conductivity, and thereafter microorganisms in the sample liquid are analyzed by dielectrophoresis (for example, see Patent Document 2).
Also a microorganism activity measuring device which, when the activity of microorganisms is to be measured, performs a rapid measurement in substantially real time to detect quantitatively and simply the activity of the microorganisms, and a microorganism activity measuring method which is used in the measurement are known. In the method, the kind of the microorganisms and the electric conductivity of a sample liquid are input, and a voltage (the amplitude and the frequency) which is optimum for measuring the activity is selected from Table 1 (for example, see Patent Document 3).
Furthermore, a measuring method is known in which, when dielectrophoresis and an impedance measurement are to be simultaneously performed, the voltage for inducing the dielectrophoresis and the voltage and frequency for performing the impedance measurement are respectively independently set. Typically, the voltage for performing the dielectrophoresis is set to 12 Vrms or higher and 1 kHz to 50 MHz, and that for performing the impedance measurement is set to 0.8 Vrms and 800 Hz (see Non-patent Document 3).
TABLE 1OPTIMUM FREQUENCYCONDUCTIVITYFOR MEASURINGOF DEGREE OF NAME OF MICROORGANISMSUSPENSIONVOLTAGEACTIVITYESCHERICHIA COLI0.1 mS/m3 Vpp 1 MHzPSEUDOMONAS AERUGINOSA0.1 mS/m3 Vpp 5 MHzKLEBSIELLA PNEUMONIAE0.1 mS/m3 Vpp10 MHz